In essence, the project has deviated little from the plan outlined in the original proposal. We still hope to elucidate mechanisms whereby lymphocytes kill some, but not all, tumor (or undifferentiated) cells in the absence of detectable humoral antibody. During the coming year, we shall primarily emphasize studies designed to elucidate "recognition" step of the NK-target cell interaction. Since this must be a property of the target cell membrane, the plasma membranes of NK-sensitive, NK-insensitive, and PMA-treated target cells will be isolated and purified by established methods (Wang et al., Jour. of Reticuloendothelial Society, 19:333, 1976). This will involve homogenization and sucrose gradient centrifugation, after which the extracted membrane proteins will be separated on SDS-PAGE. The membrane preparations will be monitored for purity by enzyme analyses and EM. If any differences in patterns become apparent, the bands in question will be eluted and characterized by standard biochemical techniques. Another approach not described in our original proposal is the possible detection of antigenic differences between NK-sensitive and NK-insensitive cells by antisera raised to these cells. Since, as a rule, antigenicdeterminants are lost during cell differentiation, we intend to use fetal fibroblasts, which we have already shown during the past year to be sensitive to NK lysis, to raise antisera in rabbits.If our hypothesis is correct, absorption of the antiserum with adult fibroblasts should retain antibody activity to fetal fibroblasts. The antiserum will then be used to isolate the determinants which may be involved in NK cell recognition. Theoretically, this should relate to the distinguishing of a band we hope to delineate on SDS-PAGE. The remainder of the studies during the coming year will essentially be those outlined in the original proposal. For instance, we have not yet tested whether the Ia antigen plays any role in target cell recognition. We have previously postulated that Ia antigens, which are often associated with cells, could serve as an anchorage point for cytotoxic lymphocytes. Therefore, we intend to pursue studies which suggest that cells which possess this antigen may provide a substrate for NK cells. Although electron microscopic and freeze-fracture analyses on various kinds of target cells before and after PMA treatment were begun, we have not yet detected any significant morphological differences. The NK-target cell conjugates are being subjected to similar analyses. (AG)